Highly hydrated paramagnetic amorphous calcium carbonate nanoclusters as an MRI contrast agent

Amorphous calcium carbonate plays a key role as transient precursor in the early stages of biogenic calcium carbonate formation in nature. However, due to its instability in aqueous solution, there is still rare success to utilize amorphous calcium carbonate in biomedicine. Here, we report the mutual effect between paramagnetic gadolinium ions and amorphous calcium carbonate, resulting in ultrafine paramagnetic amorphous carbonate nanoclusters in the presence of both gadolinium occluded highly hydrated carbonate-like environment and poly(acrylic acid). Gadolinium is confirmed to enhance the water content in amorphous calcium carbonate, and the high water content of amorphous carbonate nanoclusters contributes to the much enhanced magnetic resonance imaging contrast efficiency compared with commercially available gadolinium-based contrast agents. Furthermore, the enhanced T1 weighted magnetic resonance imaging performance and biocompatibility of amorphous carbonate nanoclusters are further evaluated in various animals including rat, rabbit and beagle dog, in combination with promising safety in vivo. Overall, exceptionally facile mass-productive amorphous carbonate nanoclusters exhibit superb imaging performance and impressive stability, which provides a promising strategy to design magnetic resonance contrast agent.

1. Because of the amorphous state, the samples should be non-homogeneous microscopically. Therefore, there is no evidence to show how much sample is actually involved in the tests, especially those involved cells. High/low local concentrations of the samples could give a bias to the conclusion, and the reproducibility of tissue/cell-related experiments might be poor.
2. Not like other nanoclusters applied as MRI contrast agents, there is no indication of "relaxivity density" (defined as [relaxivity per unit]/[unit volume or molecular weight]) or "relaxivity per Gd" (commonly used to judge Gd-chelates). Those parameters are more specific and more applicable than an overall relaxivity value, especially for the macromolecules studied here.
3. Because the structure of ACNC is not uniform and not clearly reported, it might be difficult to propose further modifications on this material. Not like nanoparticles or complexes which can be coated or conjugated with vectors for targeted therapy/imaging, there seems little room for the amorphous materials.
4. The stability tests are not comprehensive. It is not surprising that ACNC did not leak free Gd(III) ions obviously in aqueous solution or in in vitro tests. However, there at least should be a competition test between ACNC and other Gd(III) chelators. For examples, the competition between a new Gd(III) complex and excessive DTPA free ligand is a classical way to compare the stability.
Besides, although Fig. 3G is described as results for stability tests (line 165). Its picture caption and axis labels are unclear and there is no information on how this experiment was conducted.
5. It could be difficult to precisely control the administration amount in practical uses (minimal dosage) because the Gd content in a tiny amount of amorphous materials could vary largely and then the performance will be quite different.
6. Mentioned in the manuscript is that most ACC are instable in aqueous solution which limits their applications, but the authors report ACNC to be biocompatible. It is necessary and will be impactful to explain in detail how the added Gd(III) could dramatically change the physical properties. 7. Other cell lines for MTT may be expected. Only one cell line for biocompatibility seems not convincing enough. In addition, different incubation times may also be required in this experiment.
8. For the confocal imaging ( Fig. S14 and Fig. S15, p10, line190-192), Why are the incubation times for these two experiments different? In addition, results with longer incubation time will be more convincing for this study. 9. For the clearance study, in Fig.S21, the amounts of ACNC remaining in the liver and spleen in mice after even 30 days were not negligible and could be harmful. 10. In the nanocluster characterisation part (p5), although there is no exact formula for the nanocluster, the doping percentage of gadolinium in ACNC is still expected to be mentioned for the following analysis and comparison.How will the amount of added Gd(III) salts affect the overall performance (relaxivity, toxicity, stability)? There is no discussion on it.
relaxivity of ACNC reached the highest under the condition of 'n', yet got reduced when the amount of 1 PAA was '2n' and 'n/2'. Specifically, the relaxivity decreased significantly when the amount of PAA was 2 only '10/n' (Figure 1.1b, c). 3 In light of the results, it is clear that water content per Gd acts strongly on the performance of the 4 products. These comparisons suggest that relaxivity is changing along with water content per Gd as they 5 share a similar pathway of changing.  In order to prevent possible interference which the free metal ions generated from degradation 1 precipitate with the phosphate ions in the PBS buffer, 1 mL of freshly prepared chloroazotic acid 2 (HNO3/HCl = 3:1) was added to the collected surrounding PBS or acetate solution, leading to a strong  6.8, 6.5, and 6.0) and acetate buffers (pH 5.5, 5.0, and 4.5) at 37 °C within 7 days. Based on the results as shown in Figure 1.2, although an obvious leakage of Ca ions can be observed 10 at a pH range of 6.0-6.8, the leakage of Gd ion was scarcely detected with 7 days. We presume that Gd(III) 11 within ACNC will tend to form GdPO4 with phosphate in the PBS buffer due to the lower thermodynamic  3. Arsenazo III mediated chromogenic assay was used to detection of leakage of Gd ion in this work.

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Would direct mixing of ACNC and Arsenazo III under simulative physiological environment be a 12 reasonable way to evaluate its stability? 13 ** Thanks for your valuable comments.
14 As a generally used method to detect the leakage of gadolinium ion, the absorption spectra of 15 Arsenazo III has been successfully employed in gadolinium chelates in human serum and urine [Ref. 10 aqueous solution was mixed with Gd 3+ , pink solution turned blue due to the formation of Arsenazo-Gd 3+ 20 complex (Figure 1.3a). Furthermore, the formation of arsenazo-Gd 3+ complex at low concentrations of 21 Gd ion can be characterized by the absorption peak at 658 nm in UV-Vis spectra. As shown in Figure 1.3b, 22 free gadolinium ion at 1 µg/mL was detectable by arsenazo III, while the concentration of ACNC dialysis 1 solution was about 2.5 (Gd) mg/mL. So, the limit of detection (LOD) was superior to 0.1%, which was 2 sensitive to detect the leaked gadolinium ion.  In order to assess the accuracy of spectroscopy method and its LOD, we measured quantitatively the 11 possible leakage of gadolinium ion from the ACNC using Inductively Coupled Plasma Mass Spectrometry 12 (ICP-MS), whose sensitivity against gadolinium was transparently higher than that of absorption spectra 13 of arsenazo III. 14 In serum stability research, ACNC was dispersed in human serum with a final concentration of 1 15 mmol (Gd) /L. Meanwhile, to simulate the elevated phosphate concentrations in serum in patients with 16 end-stage renal disease, the same concentration of ACNC was dispersed in human serum supplemented 17 an additional phosphate concentration of 10 mmol/L.

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The co-incubated serum samples were loaded into dialysis bags with the MWCO of 1000 at different 1 time points during 15 days, then the dialysis bags were immersed into PBS and PBS with an additional 2 phosphate concentration of 10 mmol/L for 1 day at 37.°C. In summary, no characteristic absorption 3 correspoding to arsenazo-Gd 3+ complex was detected in two kinds of serum samples at any time ( Figure   4 1.4). This study was a valid evaluation to investigate the ACNC stability in serum at normal and elevated       4. The authors claim that ACNC can be effectively cleared by the kidneys. However, Fig.S21 shows that 1 Gd3+ is mainly distributed in the liver. Even after 1 day and 7 days, the content of Gd3+ in the liver is 2 much higher than other organs. The author should explain the reason for this phenomenon. Gd3+    Furthermore, nanoparticle size is the key factors affecting filtration efficiency, as glomerular filtration 14 slits in the kidneys are responsible for filtration efficiency. Since the threshold for filtration size of the 15 glomerular capillary wall is usually 6-8 nm (5.5 nm for metal-based nanoparticles), decreasing the particle 16 size of inorganic nanoparticles is the first step in improving renal clearance of these particles [Ref. 16.  In the biodistribution study mentioned in our previous manuscript (Figure 1.6a), a noticeable 19 distribution of Gd element in liver was observed. The main reason for this result is that ACNC was 20 administered with simulated bolus injections (~1 mL/s) to mimic the rapid clinical medication of MR 21 contrast agent via high-pressure syringe that was administrated in our in vivo studies. ACNC would form 22 aggregates with larger size due to this injection method, which is difficult to quickly clear from the kidney.           (Figure 1.7). According to these standard data and the 11 measured indices in control group, there was no obvious difference in the blood biochemical indexes in 12 experimental groups with i.v. injection of ACNC (5 mg/mL). Moreover, according to this valuable 13 suggestion and comment, we supplied further compatibility evaluation of ACNC on beagle dog ( Figure   14 1.8). This dosage (9 mg/kg) is three times higher than that for MRA. These data were supplied in this 15 revised manuscript.  Meanwhile, the reference normal range from 30 healthy Balb/c mice in our group was marked by pink.   In this work, the authors developed ultrafine paramagnetic amorphous carbonate nanoclusters (ACNC) in 2 the presence of both gadolinium occluded highly hydrated ACC-like environment and poly(acrylic acid).        We have already added the information in the captions based on your suggestion.  ** Thank you for this valuable comment. 16 We have already supplied the information based on your suggestion, which improved the quality of our     ** Thank you for this valuable comment. 20 In line with your suggestion, semiquantitative analysis was performed using ImageJ software. It is 1 clearly displayed that signal intensities in ACNC groups have a remarkable enhancement in comparison 2 to those in Gd-DTPA groups. The results of semiquantitative analysis for each graph were added to the 3 revised paper (Figure 2.6-2.11).     To avoid the potential risk, renal clearance was the more desirable and preferred excretion route for   Herein, we conducted a pilot research on the clearance of our ACNC by MR imaging and analysis of 8 the urine from rat, because it is difficult to collect the whole urine from the large animal beagle dog or 9 small mouse. The urines were collected using metabolic cages (Figure 2.14a). In the urine collected 24 10 hours after the intravenous injection of ACNC, the content of gadolinium was detected by ICP-AES, 11 indicating the renal clearance efficiency of approximately 13% ID at 24h (Figure 2.14b). The renal 12 clearance efficiency at 24 h post injection in 5 rats was $13.37%ID, $11.69%ID, $12.49%ID, 13 $13.84%ID, $14.34%ID, respectively. Of course, more systematic researches are necessary to confirm 14 the renal clearance of ACNC in other animals, which is still ongoing.     The authors reported interesting finding for potential MRI agents, however, there are some important 2 issues, especially biological safety, I don't think this manuscript can be published.  The amorphous state refers to x-ray amorphous, which means that the material does not have a long 12 range order, which can be detected by X-rays. This does not mean that the samples are microscopically   In our previous studies, we have obtained the molecular weight of ACNC by the use of AUC 6 measurements ( Table 1). We dissolved a weighted amount of ACNC lyophilized powder with freshly  Table 2. Compared with some Gd-based nanoparticles and 8 macromolecules that with relaxivity density already known, ACNC performed excellent in terms of 9 relaxivity density.   purified via centrifugation (Figure 3.2a, b). Fluorescence spectra of BSA-FITC, ACNC, and ACNC-BSA-

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FITC were detected to characterize whether BSA-FITC could be modified on the surface of ACNC. As 22 33 shown in Figure 3.2c, BSA-FITC has a characteristic absorption at ~550 nm, and appearance of the 1 characteristic peak of ACNC-BSA-FITC confirmed the success of modification.
2 Then, we attempted to load drug on ACNC using doxorubicin hydrochloride (DOX). ACNC-Dox 3 was prepared by simply mixing ACNC with Dox, purifying by centrifugation, and washing with deionized 4 water (Figure 3.2d, e). Figure 3.2f shows UV-Visible absorbance spectra of DOX, ACNC and ACNC-5 DOX. DOX presented a characteristic absorption peak at around 480 nm, and the peak was retained after 6 DOX being loaded onto ACNC. Overall, the successful modification of protein and drug serves as strong 7 supporting evidence indicating that ACNC could be a promising type of nanoagent with great potential 8 for theranostics.   ** Thank you for this valuable comment. 14 We addressed this issue by applying DTPA in the ligand competition assay. 1 mL ACNC aqueous 15 solution (5 mM Gd) was added into 1 mL DTPA solution (5 mM) and the homogeneous solution was taken 16 for measurement. Compared with the commercial Gd-DTPA, the concentration of Gd-DTPA solutions was 17 identical to ACNC solutions as 5 mM Gd. 18 In addition, to assess the relaxivity stability of ACNC, the longitudinal relaxation time was monitored.

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There was no observable evidence showing that relaxivity of ACNC was significantly altered, which 20 confirmed that ACNC was not affected by the competition between Gd(III) complex and excessive DTPA 21 free ligand (Figure 3.3).     5. It could be difficult to precisely control the administration amount in practical uses (minimal dosage) 10 because the Gd content in a tiny amount of amorphous materials could vary largely and then the 11 performance will be quite different.

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** Thank you for this valuable comment.

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In practice, the amount of Gd ions was quantitatively determined by inductive coupled plasma atomic 14 emission spectrometer (ICP-AES). The detection limit of ICP-AES was 0.001 ,g/mL. ACNC acts 15 essentially on the application of this method as a contributor by delivering favorable dispersity and stability. 16 The commercial Gd-DTPA used as a control subject can also be precisely measured by this method. To  applications, but the authors report ACNC to be biocompatible. It is necessary and will be impactful to 7 explain in detail how the added Gd(III) could dramatically change the physical properties. 8 ** Thank you for this valuable comment. 9 This suggestion is very useful for us to investigate the stability of Gd occluded ACC complex. 10 According to this suggestion, the transformations of ACC-Gd were investigated in different mediums 11 (ethanol, water) to further explore how Gd affected the stability of ACC (Figure 3.6). Gd was stable in ethanol over 6 months.

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(2) After precipitates were further washed twice by water, both products were dispersed in water. ACC 3 transformed to calcite within one day. As a thermodynamically stable crystalline form, the calcite phase 4 would be maintained over the course of the next month. In comparison, the fresh-prepared ACC-Gd could 5 maintain amorphous phase for tens of minutes. Overall, Gd should play a reasonable role in retarding the crystallization of ACC. 9 Although a crystalline phase was observed in ACC-Gd dispersed in water, there was still higher water 10 content was held. However, in comparison with ACC-Gd, the water in ACC was rarely held. According to 11 the TGA result, the weight loss before 300 o C could be calculated as water content (Figure 3.7). The 12 weight loss of ACC-Gd and ACC powder exposed to dry air over 6 months was more than 10% and only 13 0.1%, respectively. Note that ACC, which was precipitated in a hydrated form has completely lost its 14 hydration water under these conditions. 42 Figure 3.7 Thermogravimetric curve of ACC and ACC-Gd lyophilized powder isolated by deionized 1 water and then exposed to dry air for over 6 months. The weight loss before 300 o C could be attributed to 2 the loss of water.

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In the isolation process of ACC composites, both additive-free ACC and ACC-Gd were washed by 5 ethanol. The changes of morphology of ACC and ACC-Gd after dispersed in ethanol in two weeks were 6 observed by TEM (Figure 3.8). A disordered emulsion-like structure was formed initially in additive-free 7 ACC, as well as in ACC-Gd with smaller particle size. For ACC, branched aggregates with large particles 8 were occurred 3 days later and several micrometers sized crystals could be observed one week later, 9 indicating the transformation from small ACC nanoparticles into thermodynamically stable form and 10 further ripening. Two weeks later, few small nanoparticles were retained and larger crystals were formed.

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In contrast, no obvious growth and aggregate of particle was observed in ACC-Gd. in water, and a calcite phase could be detected by XRD analysis (Figure 3.6). However, when ACC-Gd 6 was dispersed into aqueous solution, it would disperse well at first. Then, an aggregation of ACC-Gd could 7 be observed in the next 2 hours to form a gel-like flocculation without any sediment. The changes of 8 morphology of ACC and ACC-Gd dispersed in water for a long time were also observed by TEM ( Figure   9 3.10). In the TEM images, few emulsion-like ACC could be found in ACC dispersed in water, and micro-      In line with your suggestion, HK-2 cells were cultured for 48 hours to assess the cytoxicity including 9 additional markers (mitochondrial, nuclear DNA and cellular proliferation). Gd-DTPA with identical 10 concentrations served as control.

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To determine whether damage of nuclear DNA occurred, we used TdT-mediated dUTP Nick-End

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Labeling (TUNEL) method to examine apoptotic cells. Apoptotic cells were labeled by red fluorescent 13 probe Cyanine 3 (Cy3). The results from the immunofluorescent TUNEL staining assay of HK-2 ( Figure   14 3.13) showed that ACNC did not cause DNA damage even at a high concentration of 500 ,g (Gd)/mL.    9. For the clearance study, in Fig.S21, the amounts of ACNC remaining in the liver and spleen in mice 7 after even 30 days were not negligible and could be harmful. 8 ** Thank you for this valuable comment. 9 Previous studies reported that the majority of intravenously administered nanoparticles is removed    16 In the biodistribution study mentioned in our previous manuscript (Figure 3.14a) aggregates with larger size due to this injection method, which is difficult to quickly clear from the kidney.

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Given this, we performed another pilot evaluation of ACNC biodistribution in vivo with a relatively slow 22 injection speed (~500 ,L/s). The result showed that intravenous injection with lower speed could 1 significantly decrease the accumulation of ACNC in the liver and spleen, which provides valuable guiding 2 experience for the pre-clinical administration (Figure 3.14b).  In addition to the particle size, surface modification also plays a role in affecting the renal clearance 8 of engineered nanoparticles. Evidence has shown that nanoparticles with polyethylene glycol (PEG) 9 modification can significantly lower the adsorption of serum protein and thereby retard the NMs uptake  There will be cell type-specific responses to the accumulation of NMs in the liver and their uptake 1 by liver cells. As more effort is put into understanding nano-liver interactions, we could expect to develop   10. In the nanocluster characterisation part (p5), although there is no exact formula for the nanocluster, 21 the doping percentage of gadolinium in ACNC is still expected to be mentioned for the following analysis 22 58 and comparison. How will the amount of added Gd(III) salts affect the overall performance (relaxivity, 1 toxicity, stability)? There is no discussion on it.
2 ** Thank you for this valuable comment.

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As you suggested, the extra study of Gd helped us better evaluate the performance and stability of 4 ACNC. The amount of substance ratio of Gd and Ca was calculated by measuring the element masses 5 using ICP-AES method. According to our calculations, the Gd/Ca atomic ratio of ACNC was 6 approximately 1:3, suggesting the doping percentage of Gd 3+ in ACNC was to be around 25%. 7 We further designed and fabricated two more amorphous carbonate nanoclusters with varying ratios 8 of Gd added for comparison purpose. In the synthesis of ACNC, mol[GdCl3]: mol[CaCl2] equals to 1:5. 9 We also prepared two samples with initial Gd/Ca feed ratio as 1:10 and 1:2 (denoted as ACNC(Gd/Ca=1:10) 10 and ACNC(Gd/Ca=1:2), respectively).  We conducted MTT assay in order to examine the cytotoxicity of these two products. Both HK-2 and 5 HaCat cells were incubated with ACNC(Gd/Ca=1:10) and ACNC(Gd/Ca=1:2) individually for 24 and 48 hours.

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When the amount of Gd was increased to 500 ,g (Gd)/mL, cell viability still remained above 98%,  The relaxation times of these products were also measured using a 3.0 Tesla MR scanner. The results 5 showed that ACNC(Gd/Ca=1:10) retained a good MR performance with high relaxivity as 34.25 mM "1 ·s "1 6 when the proportion of doping Gd was low. However, when the level of incorporation of Gd was elevated, 7 the longitudinal relaxivity (r1) values of ACNC(Gd/Ca=1:2) decreased remarkably as 17.21 mM "1 ·s "1 ( Figure   8 3.17). One explanation could be that the excessive amount of Gd may potentially perturb the highly 9 hydrated ACC-like environment, which in turn affected the generation of ACC with high water content. 10 As mentioned in our previous manuscript, the relaxivity of AGC-PAA was as low as 7.91 mM -1 ·s -1 if only 11 61 Gd elements were used to form amorphous nanoclusters. In summary, the doping amount of Gd 1 significantly affected the relaxivity of paramagnetic ACNC. After comparing two ACNC products with 2 different Gd doping amounts (ACNC(Gd/Ca=1:10) and ACNC(Gd/Ca=1:2)), ACNC reported in our original 3 manuscript (the initial Gd/Ca feed ratio at 1:5, and a final Gd/Ca atomic ratio at 1:3) was found to be the 4 best product in terms of contrast performance.